THERAPEUTIC USE OF THE ß2m PROTEIN

ABSTRACT

The use of beta2-microglobulin (β2m) as active ingredient, in particular in pharmaceutical compositions intended for the treatment of autoimmune diseases.

The present patent application concerns the medical field, in particularthat of the treatment of autoimmune diseases.

The invention relates more particularly to the use of thebeta2-microglobulin protein (β2m) as active ingredient, in particular inpharmaceutical compositions intended for the treatment of autoimmunediseases, such as, for example, multiple sclerosis or Crohn's disease.

Preamble

The β2m protein is a protein having an average molecular weight ofapproximately 11.6 kDa, generally formed from 99 amino acids, whichenter into the constitution of the major histocompatibility complex (MHCI or HLA I) [Cunningham B. A. et al. The complete amino acid sequence ofbeta-2-microglobulin (1973) Biochemistry 12: 4811-4821].

It is to be recalled that the MHC I histocompatibility complex plays acentral role in the recognition of “self” and “not-self” by the immunesystem. These complexes are present on the surface of most human cells,with the exception of the erythrocytes. On their surface they present ahigh number of antigens on the basis of which the T lymphocytes (CD8)are capable of discriminating the cells of an individual from the cellsthat are foreign thereto, diseased or undergoing a tumor transformationprocess.

The MHC I complexes are composed of a glycosylated heavy chain (HC), ofapproximately 44 kDa, and of a light chain, the β2m, which associatesnon-covalently with the extracellular domain of the heavy chain. The αchain of MHC I is composed of three extracellular domains, (α1, α2 andα3) and of a transmembrane segment as indicated in FIG. 1A. The β2massociates with a sequence of amino acids situated in the zone where theend of the al domain and the start of the α3 domain in the HC are inproximity [Gussov, D. et al. (1987), The human beta-2-microglobulingene: primary structure and definition of transcriptional unit (1987)Journal of Immunology 139:3132-3138]. The genes coding for the MHC Imolecules have been numbered in the order of their discovery and classedinto groups (A, B and C) and complexes (D, H and G).

The antigen-presenting cells (APCs) use the complexes of MHC I type asantigen presenters to T-cells (CD8) of the immune system. The antigenspresented by MHC I are generally constituted by a variety ofpolypeptides having 8 to 10 amino acids, which results from thesplitting of endogenous proteins by the proteasome. These antigens areloaded onto the peptide cavities present on the surface of the sub-units(HC and β2m) of the MHC I complexes during their formation within theendoplasmic reticulum. Once the antigens have been loaded, the MHCcomplexes are exported to the surface of the cell. The anchoring of theMHC I complex to the plasma membrane is then provided by thetransmembrane domain of the heavy chain situated at the α3 domain.

The subunit formed by the β2m protein is distinguished from the heavychains in that the sequence is practically invariable and in that itspolypeptide chain is not glycosylated.

Even if its physiological role has not yet been fully elucidated, it hasbeen shown that the β2m protein plays a dominant role with regard to theother protein forming the MHC I complexes, on the one hand, uponassembly of the MHC I/antigen complex [Androlewicz M J., et al. (1994)MHC class I/β2-microglobulin complexes associate with TAP transportersbefore peptide binding, Nature 368, 864-867], in which the β2m proteinspecifically binds to the TAP-1 protein, [Corr et al. 1992. Endogenouspeptides of a soluble major histocompatibility complex class I moleculeH-2Lds: sequence motif, quantitative binding and molecular modeling ofthe complex, JEM, 176(6):1981-92], making it possible to ensure that theconformation of the antigen binding site is maintained [Ljunggren, H-G.(1990) Empty MHC class I molecules come out in the cold. Nature. 346,476-480] and, on the other hand, when the MHC I/antigen complex isexported to the cell surface. The β2m is involved in the folding of theheavy chain and is also found to be involved in the presentation of theantigen to the T-cells (CD8). The β2m also contributes to the stabilityof the MHC-I/antigen complex [Neefjes, F. F. et al. (1993) Selective andATP-dependent translocation of peptides by the MHC-encoded transporter.Science. 261 (5122): 769-771].

Transgenic animals lacking any β2m prove viable, but present a weakenedimmune response, making them more susceptible to viral and parasiticinfections. The reduction in the immune response in these animalsappears to be correlated with the fact that their cells present very fewantigens with regard to their MHC I complex and that the majority oftheir T lymphocytes are not functional [Pereira P., et al. (1992)Blockade of transgenic gamma delta T cell development in beta2-microglobulin deficient mice, EMBO Journal 11:25-31].

The β2m protein is also described as being involved in the glycosylationof heavy chains in the Golgi apparatus [Sege et al. (1981) Role ofbeta2-microglobulin in the intracellular processing of HLA antigens.Biochemistry. 20 (16), pp 4523-4530].

The β2m protein is also involved in other phenomena such as theregulation of intercellular signaling and the correct folding of keyproteins, such as HFE (Human hemochromatosis protein) which regulatesthe flow of iron in the cell.

It has also been established since the end of the 1980's that β2m mayfavorably improve the antigen response and be used as a vaccine adjuvantto stimulate the immune response linked to the T lymphocytes (CD8).

Numerous documents indicate that β2m may thus be incorporated intovaccine compositions in combination with molecules having the task ofinducing an immune reaction, such as specific virus or tumor antigens.

In such vaccine compositions, β2m may be present in different forms,purified or recombinant. Genetic constructions have thus been describedin which the gene coding for β2m is fused with genetic sequences codingfor immunogenic peptides with the aim of expressing fusion proteinsintended to elicit a specific immune reaction in-vivo [WO 99/64957].

By itself, the β2m protein is very weakly immunogenic, since it is notglycosylated. On account of this, in the above vaccine compositions, β2mis always used as an adjuvant and not as an active ingredient.

This is doubtless due to the fact that, to date, no therapeutic effectof β2m has been observed capable of justifying its use in pharmaceuticalcompositions.

Apart from in vaccination, certain prior art documents indicateinactivated or modified forms of the β2m protein in therapeuticcompositions.

The international application WO 02/102840 thus describes a β2m renderednon-functional intended to form inactive MHC I complexes, which can nolonger activate the CD8 T lymphocytes. The MHC complexes so formed areused as a “lure” for the immune system with the object of obtaining animmunosuppressant effect.

Another international application WO 02/24929 describes therapeuticcompositions in which the β2m is conjugated to the HFE protein as avector, to deliver drugs (active ingredients of those compositions) tothe intracellular compartment.

It should be noted that in these types of applications, the β2m proteinis not used in its wild-type functional form as active ingredient, butas a pharmaceutical support or vector, in the presence of activeingredients not directed to the MHC.

Moreover, in contrast to any therapeutic application, the β2m protein isoften used as a marker for different pathologies, in particular as ameans of diagnosis.

Thus, the immune deficiency syndrome in the AIDS disease, which mayreveal itself many years after the infection with HIV, is preceded by anabrupt increase of the β2m concentration in the blood.

Certain publications [Wu C. H. et al. (2001) Oncogene 20:7006-20] stressthat the increase in the β2m concentration correlates with and isperhaps involved with the development of certain cancers, in particularbone cancer and prostate cancer [Gross M. et al. (2007) Clin. CancerRes., 13:1979-1986]. For other cancers, drops in the β2m serumconcentration are observed, as in cancer of the colon [Kaklamanis L. etal. (1992) Int. J. Cancer 57:379-385].

The dosage of β2m in the blood (and more particularly the blood serum),the cerebrospinal fluid or the saliva, is frequently used in thediagnosis of certain infectious or parasitic diseases but also,primarily, for the diagnosis of certain diseases of the kidney, of thelymphatic system, rheumatism, inflammatory diseases, and neurologicaldiseases such as Alzheimer's and frontotemporal dementia [Davidsson P.et al. (2002), Proteome analysis of cerebrospinal fluid proteins inAlzheimer patients Clinical Neuroscience and Pathology 13: 611-615;Hansson S. F. et al. (2004), Validation of a prefractionation methodfollowed by two-dimensional electrophoresis-Applied to cerebrospinalfluid protein from frontotemporal dementia patients Proteome Science2:1-11].

In persons considered to be in good health, the average concentration ofβ2m in the blood remains relatively constant, less than or equal to 2mg/l, which is not the case in the above-mentioned diseases, in whichthat concentration may attain values as high as 4.0 mg/l.

For certain pathologies, the increase in serum β2m could be caused byincreased “shedding” (release of cell surface proteins) of the β2m[Bellotti V., et al. (1999) Cell. Mol. Life. Sci., 55-977-991].

The plasma β2m circulating in the blood is normally filtered in thekidneys by the glomeruli, then reabsorbed and catabolized in thetubules.

Studies have shown that half the plasma β2m (free form of the β2m),which is renewed each day, comes more particularly from the recycling ofthe MHC-I complexes. This renewal by itself thus appears to contribute ahigh production of serum β2m of approximately 150 mg/24 h for a personof average size. However, the “turn-over” would appear to stabilize theserum concentration at 2 mg/l.

In patients under dialysis, for whom β2m is not eliminated by thekidneys, the accumulation of β2m in the body fluids has deleteriousconsequences. In particular, it induces arthropathies and neuropathiesby formation of amyloid plaques in certain connective tissues (nervousand articular) [Ohshi K., et al. Pathogenesis of beta2-microglobulinamyloidosis (2001) Pathol. Int. 51:1-10].

In osteoarthritis (arthrosis), β2m is described as having an inhibitingeffect on the proliferation of the chondrocytes, a consequence of whichis to accentuate the destruction of the cartilages [WO 2004/020586].

In the case of certain autoimmune diseases, such as multiple sclerosis(MS), it is common to monitor changes in the concentration of β2m inpatients, to anticipate the onset of inflammatory episodes [Bagnato, F.,(2003), beta-2 microglobulin and neopterin as markers of diseaseactivity in multiple sclerosis Neurol. Sci. 24:51301-51304]. Theconcentration of β2m is then preferably measured in the cerebrospinalfluid since the concentration of β2m in the blood is considered as toovariable [Caudie C. et al. (2005), Valeurs usuelles et utilitédiagnostique de la β2-microglobuline dans le liquide céphalorachidienAnn. Biol. Clin. 63(6):631-637; Ryu O. H., et al. (2006) Rheumatology,45:1077-1086].

The involvement of β2m in autoimmune diseases remains unclear and wouldmerit further study.

The autoimmune diseases form a large set of diseases the symptoms ofwhich may be attributed to hyperactivity of the immune system, with thepresence or absence of autoantibodies, directed against substances ortissues which are normally present in the body.

It is certain that the immune response against “self” in autoimmunediseases results from the activation of the T lymphocytes via the MHCsystem and several mechanisms may cause this.

-   -   In the immune system:        -   By induction of autoantibodies using T cells by presentation            of antigens known by said autoantibodies. This is the case            of systemic lupus erythematosus (SLE), Hashimoto's            thyroiditis, multiple sclerosis (MS), insulin dependent            diabetes (type I), etc.    -   In cells:        -   induction of an autoimmune response by activation of T cells            specific to a viral antigen;        -   Alteration with regard to APC-MHC-I/TcR (T cell receptor)            recognition and with regard to the signaling cascade(s) of            the activated T lymphocyte.        -   Improper assembly in the APC of the components of the MHC-I            system.        -   Defect(s) in the operation of the regulatory cells.    -   At molecular level:        -   Molecular mimicry or tolerance;        -   MHC I as an autoantigen;

The autoimmune diseases are generally considered to result from aconjunction of a genetic predisposition and of an infectious episodeduring which the body develops a immune reaction to its own antigens.However, the exact causes of these diseases have not been identifiedprecisely.

The most widespread autoimmune diseases are rheumatoid polyarthritis,Sjögren's syndrome, Hashimoto's thyroiditis, Addison's disease, systemiclupus erythematosus, scleroderma, fibromyalgia, myositis, ankylosingspondylitis, insulin dependent diabetes of type I, Crohn's disease,Celiac's disease and multiple sclerosis (MS).

Among what are referred to as the “orphan” diseases, there are numerousother disorders that are suspected of also being autoimmune diseases.Amyotrophic lateral sclerosis (ALS) is one of those diseases, for whichno effective treatment is currently available.

Two types of autoimmune diseases should be distinguished: the specificautoimmune diseases and the non-specific autoimmune diseases.

In the non-specific diseases, different organs are affected, causingsystemic diseases such as rheumatoid arthritis, systemic lupuserythematosus, Sjögren's syndrome and scleroderma.

The specific diseases are especially limited to certain organs. The mostcommon are insulin dependent diabetes, thyroid diseases, Addison'sdisease, a few diseases of the kidneys, of the lungs, of the digestivesystem, and especially multiple sclerosis.

Current therapies comprise a range of approaches fromanti-inflammatories to immunosuppressants through antimetabolites andanti-cancer drugs. By way of example the following are used:non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids,antimetabolites (methotrexate, azathioprine), cyclophosphamide,sulfasalazine, gold salts, cyclosporin A, mycophenolate, andleflunomide.

Recently, interferon β has been recommended for MS and the derivativesof chloroquine (used against malaria) are recommended for the treatmentof lupus erythematosus and of rheumatoid polyarthritis.

These treatments, used for treating other diseases, are not very welladapted and have numerous undesirable side effects, in particular whenthey are used over the long-term. Moreover, although they may enable thesymptoms of those diseases to be at least partially attenuated, they donot enable remission of the diseases to be obtained.

The inventors designated by the present application took a particularinterest in the situation of four patients affected by apparentlydistinct autoimmune diseases:

-   -   A first patient (PI) suffering from a non-specific autoimmune        disease, that is to say not affecting a specific organ, but also        suffering from Hashimoto's thyroiditis and primary Sjögren's        syndrome;    -   A second patient (P2) suffering from MS, originally, with        duodenal lymphocytic infiltration subsequently;    -   A third patient (P3) suffering from Celiac's disease;    -   A fourth patient (P4) suffering from Hashimoto's thyroiditis and        from Celiac's disease.

For these patients, the inventors sought to establish the ratio of thequantities HC (MHC-ABC)/β2m coming from the lymphocytes, isolated fromthe blood of the patients using the conventional methods indicatedlater.

Surprisingly, this HC/β2m ratio proved to be on the increase in thesefour patients, compared with that of control donors, whereas there serumβ2m concentration was average: approximately 1.9 mg/l for P1, 1.8 mg/lfor P2, 1.1 mg/l for P3 and 1.1 mg/l for P4. (cf. table I).

TABLE 1 Determination of the different forms of β2m in patientssuffering from autoimmune diseases Serum β2m HC/β2m HC/β2m Patients (a)proteins (b) membranes (c) P1 1.9 1.3 1.8 P2 1.8 1.1 1.7 P3 1.1 1.6 1.5P4 1.1 1.2 2.1 HC: heavy chains of the MHC I (a) Concentration of β2m inmg/l; (b) HC/β2m calculated from the total lymphocyte proteins. (c)HC/β2m calculated on the plasma membranes isolated from a purifiedlymphocyte fraction.

The results of table 1 above show an imbalance in the HC/β2m ratio.These results have revealed an unexpected situation, whereby the MHC-Imembrane complexes present in those four patients is apparentlysignificantly deficient in β2m relative to the HC concentration, withoutthis increasing the concentration of free β2m in the blood.

These observations are to be compared to the controls in good health,who show a HC/β2m ratio in the neighborhood of 1. By contrast, β2mappears to be sequestrated in the intracellular compartment in thepatients affected by the autoimmune diseases.

These results surprised the inventors and led them to formulate thehypothesis that the four autoimmune diseases affecting the patientscould have a deficit in β2m in the membrane MHC-I complexes as a commonorigin. More generally, an HC/β2m imbalance in the MHC complexes wouldappear to contribute to the appearance of the disorders encountered innumerous autoimmune diseases.

According to this hypothesis set out later, the autoimmune reaction, inthe context of the pathologies from which four patients suffer, is notapparently the consequence of a general increase of free β2m in theblood, but on the contrary, originates with a local β2m deficit in themembrane MHC-I complexes, which is liable to alter the presentation ofthe antigens to the T cells (CD8).

It should be noted that this hypothesis in no way excludes theinvolvement of the β2m in the activation of the T lymphocytes and in theinflammatory process, as it may have been described in the prior art.

Given these first observations, the inventors carried out the analysisof HC/β2m in the total lymphocyte proteins present in other patientssuffering from MS or Crohn's disease, and were able to find that theHC/β2m ratio coming from the lymphocytes of these patients was alsogreater than of control patients.

On the basis of these results, the inventors have developedpharmaceutical compositions of which the main active ingredient is theβ2m protein in a functional form.

The purpose of these compositions is to mitigate a deficit in β2m in themembrane MHC-I complexes in patients affected by autoimmune diseases.

FIG. 1: Diagrammatic representation of an MHC complex of type I in aplane (A) and in space (B). The heavy chain (HC) is constituted by 3extracellular domains (α1, α2 and α3) and one transmembrane domain. Thelight chain (β2m), which is extracellular, inserts between the membraneand the location where the α1 and α3 of the heavy chain are inproximity. Figure B shows the position of the peptides (antigens)presented by the heavy chain.

FIG. 2: Photograph (×630) of lymphocytes placed in contact withliposomes in accordance with the invention. The liposomes have beenprepared according to the dialysis method described in Example 2. Theliposomes (light spots) are adsorbed on the membrane of the lymphocytes(HLA-ABC). The cell nucleus is intact (gray).

FIG. 3: Photograph (×630) of lymphocytes placed in contact withliposomes in accordance with the invention containing albumin. Theprotein (albumin) is rendered fluorescent with DAPI. It forms darkerspots, detected by immunofluorescence, penetrating the lighterlymphocytes (HLA-ABC positive).

FIG. 4: Photograph (×630) of liposomes prepared using the extrusionmethod (green) and containing fluorescent β2m (TRITC). A: Fluorescenceemitted by the fluorescent lipid NBD-PC-Oleyl contained in theliposomes. B: Fluorescence emitted by the fluorophor (rhodamine βisothiocyanate) coupled to the β2m. C: Superposition of the twofluorescences A and B.

FIG. 5: Photograph (×630) of lymphocytes purified from a patient (P1)and incubated with liposomes prepared according to the extrusion methodand containing fluorescent β2m (TRITC). A: fluorescence emitted by theHoechst 33342 marker which colors the nuclei of the lymphocytes in blue.B: Fluorescence emitted by the fluorophor (rhodamine β isothiocyanate)coupled to the β2m. This marking shows the incorporation of β2m into thelymphocytes that have become red in color. C: Fluorescence emitted bythe green fluorescent lipid NBD-PC-Oleyl contained in the liposomes.This marking shows the association of the liposomes with thelymphocytes. D: superposition of the B and C marking (yellowcolor)—Scale bar: 10 μm.

FIG. 6: Microscopic examination by fluorescence of liposomes containingalbumin (TRITC) after 30 days storage at two different temperatures (25°C. and 37° C.). Through observation, major differences between thedifferent types of preparation and storage cannot be distinguished. Aand B: Batch 30 (30 mg prot./150 ml). C and D: Batch 60 (60 mg prot./150ml). Bottom: Fluorescence emitted by the fluorescent lipid NBD-PC-Oleylcontained in the liposomes. Top: Fluorescence emitted by the fluorophor(rhodamine β isothiocyanate) coupled to the albumin. Scale bar: 200 nm.Enlargement ×630.

FIG. 7: Size distribution (%) of the liposomes (<50 nm, between 50 and100 nm, >100 nm) containing albumin according to time (2, 30 and 60days) and storage temperature (25° C. and 37° C.). A and B: Batch 30 (30mg prot./150 ml), C and D: Batch 60 (30 mg prot./150 ml).

FIG. 8: Size distribution (%) of the liposomes (<50 nm, between 50 and100 nm, >100 nm) containing a high concentration of β2m (Batch 80 mgprot./150 ml) according to time stored at 25° C. for 6 and 40 days.

FIG. 9: Degradation profiles for the pure or liposome-coated β2m by seraof patients or healthy donors over time. A and B: pure/liposomepreparation of β2m (serum patient 1: 51-year old woman, suffering fromHashimoto's disease). C and D: pure/liposome preparation of β2m (serumpatient 2: 73-year old woman, rheumatoid polyarthritis). E and F:control patient, healthy 62-year old man.

FIG. 10: Electrophoresis gel of protein showing the association betweenβ2m (liposome preparation) and the heavy chains of MHC-I on the cellsurface of lymphocytes purified from patients. A: In the presence ofglutaraldehyde, the HLA-β2m complexes are viewed at 55 kDa and the freeβ2m at 12 kDa. The band at 12 kDa on the track without glutaraldehyderepresents the cellular β2m (lane 3). B: Quantifying the membraneexpression of the β2m. This control makes it possible to validate theuse of glutaraldehyde for the preparation of the HLA-β2m complexes. C:Comparison of the lymphocytes from a patient suffering from multiplesclerosis (woman, 39 years old) and from a healthy donor (man, 67 yearsold) after incubation with a liposome preparation of β2m for 90 minutes.The β2m contained in the liposomes binds more on the lymphocytes comingfrom the patient (in the presence of glutaraldehyde) than it does incontrol.

FIG. 11: “In vitro” toxicity assays of free β2m or in liposomes on humanhepatic and renal cells.

A, C and E: assays on HH hepatic cells after 24, 48 and 72 hours ofexposure. B, D and F: HREpic renal cells after 24, 48 and 72 hours ofexposure. 1. control. 2. control and non-loaded liposomes. 3.3 μg freeβ2m. 4.3 μg β2m in liposome form (batch 66 μg/150 ml). 5.6 μg free β2m.6.6 μg β2m in liposome form (batch 132 μg/150 ml). Total protein contentwas estimated by the BCA method.

FIG. 12: “In vitro” toxicity assays of free β2m or in liposomes on humanhepatic and renal cells. Same labels as in FIG. 11. Result of MTT assaysfor viability.

DETAILED DESCRIPTION

The present invention thus relates to a use of the β2m protein as activeingredient, in particular for the preparation of a medicament.

The β2m protein is preferably the human form of the protein, purified orrecombinant, of which a reference polypeptide sequence as well as thegenetic determinants are described in the GENEBANK database, under theaccession number CAG33347.

If it is purified, the β2m may be obtained from the sera of healthydonors.

It may also be envisaged to have recourse to chemical synthesis sincethe protein may be used in a non-glycosylated form.

The present therapeutic use of β2m extends to the functional variants ofthat protein, that is to say to its isoforms, to mutated copies or tofragments of that protein, characterized in that they have the samefunctionality as the wild-type protein, that is to say the sametherapeutic effect as described in the present application, it beingpossible however for that effect to be reduced or increased in itsintensity relative to said wild-type protein.

Functional variant more particularly designates a polypeptide capable ofassociating with the MHC complexes present on the surface of cells, thepolypeptide sequence of which is at least 70%, preferably at least 80%,more preferably at least 90%, and still more preferably at least 95%,identical to the polypeptide sequence of the human β2m protein (thecomparison of the sequence being made, for example, using the ClustalWsoftware application).

A functional variant of the β2m preferably consists of a fragment of theβ2m protein, presenting the same therapeutic effect, or even the samebiological activity.

Such functional variants may also result from the expression ofnucleotide sequences cloned in an expression vector or in a gene therapyvector.

Numerous publications describe, for example, the presence of isoforms ofβ2m in rodents [Coding J. W. and Walker I. D. Allelic forms ofβ2-microglobulin in mouse (1980) Proc. Natl. Acad. Sci. USA 77:7395-7399] and in man [Davidsson P. et al., Proteome analysis ofcerebrospinal fluid proteins in Alzheimer patients (2002) ClinicalNeuroscience and Pathology 13: 611-615; Hansson S. F. et al., Validationof a prefractionation method followed by two-dimensionalelectrophoresis-Applied to cerebrospinal fluid protein fromfrontotemporal dementia patients (2004) Proteome Science 2:1-11]. Theseisoforms, which are distinguished more particularly by a differentisoelectric point (pI), are considered as functional variants of β2m.

Such functional variants may have certain advantages in terms of theeffectiveness of the product or its formulation relative to the purifiedhuman protein (solubility, greater stability, reduced proteolyticdegradation).

The present invention concerns pharmaceutical compositions comprisingβ2m or one of the functional variants of β2m, as active ingredient.

Preferably, the β2m or its functional variant forms the sole activeingredient of said compositions.

Within the meaning of the present invention, an active ingredient is asubstance which enters into the composition of a medicament and which isresponsible for the pharmacodynamic or therapeutic properties thereof.An adjuvant is not considered as an active ingredient within the meaningof the present invention.

More preferably, the invention relates to a pharmaceutical compositionconsisting of β2m or a functional variant of β2m contained in apharmaceutically acceptable carrier or vehicle, said pharmaceuticallyacceptable carrier or vehicle preferably being a liposome.

According to a preferred aspect of the invention, the β2m isadministered alone with said pharmaceutically acceptable carrier, or aphysiological solution, in accordance with the regulatoryrecommendations and requirements.

According to the invention, the β2m is more particularly used for itscapacity to restore a normal HC/β2m ratio within the membrane MHC-Icomplexes in a patient.

The HC/β2m ratio is preferably treated with regard to the lymphocytes,in particular the B cells. The HC/β2m ratio corresponds to the molarratio of the HC and β2m sub-units in the purified MHC I complexes.

Preferably, this ratio is returned to a level comparable to that of apatient not suffering from disease. More preferably, the β2m is usedwith the aim of reducing the HC/β2m ratio in a patient to attain a molarratio close to 1.

The present invention is more particularly directed to preventing adeficit of β2m from occurring in the MHC-I complexes in patientssuffering from autoimmune diseases.

The use of the β2m according to the invention is thus more particularlyintended for the treatment of autoimmune diseases.

The inventors have been able to determine that a deficit ofintracellular or membrane β2m could give rise to a HC/β2m ratio greaterthan 1 or even 2 in certain patients suffering from autoimmune diseases.The invention is thus directed to returning said HC/β2m to a value closeto physiological values i.e. preferably less than 2, more preferablyless than 1.5 and still more preferably less than 1.2.

The invention may of course apply to any disease linked to an imbalancein the HC/β2m ratio in the MHC I complexes, other than the autoimmunediseases.

Within the meaning of the present invention, the pathologies linked toorgan transplants or transplant rejection, are not considered asautoimmune diseases, nor as diseases caused by a defect in recognitionof the “non-self” by the immune system. To be precise, transplantrejection is considered here as resulting from a recognition of“non-self” by the immune system, and not as a defect in recognition of“self”.

The analyses carried out by the inventors in different patients indicatethat a HC/β2m ratio, calculated on the basis of the total lymphocyteprotein, greater than 1.2 may be observed at least for the followingdiseases: rheumatoid polyarthritis, systemic lupus erythematosus,Sjögren's syndrome, scleroderma, fibromyalgia, myositis, ankylosingspondylitis, insulin dependent diabetes of type I, Hashimoto'sthyroiditis, Addison's disease, Crohn's disease, Celiac's disease,amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).Although the question is still under debate within the scientificcommunity, ALS is assimilated to an autoimmune disease, in view of theresults obtained.

The invention concerns more particularly the development of a medicamentfor increasing the ratio of blood β2m to a concentration comprisedbetween 2.5 and 12 mg/l, preferably between 3 and 8 mg/l, morepreferably between 3 and 5 mg/l, to mitigate the HC/β2m deficit of themembrane MHC-I complex.

As described below, in the experimental part of the present invention,the medicament according to the invention may consist in a liposomepreparation comprising β2m or a functional variant thereof. Theliposomes may be manufactured using different techniques known to theperson skilled in the art, such as those illustrated in the examples ofthe present application. Different lipids constituting the liposomes maybe used [Medical Application of Liposomes (1986) edited by Kunio Yagi,Japan Scientific Societies Press, Tokyo, Karger].

A preferred medicament of the invention in this respect consists of aliposome loaded with β2m.

Preferably, the β2m or a functional variant of that protein constitutethe only active ingredients contained in said liposome preparation.

It is advantageous to use a liposome according to the invention as amedicament because it enables the β2m to be protected from proteolyticattacks which may take place and because it enables the β2m to bedelivered in targeted manner to the MHC-I complexes, in particular byfusion of the liposome with the phospholipids, which constitute the cellmembranes.

According to another aspect of the invention, a gene therapy vectorcoding for the β2m or for one of its functional variants is used tosynthesize the protein in-vivo, preferably in the environment of the MHCcomplexes. Such a gene therapy vector may be contained in liposomes.

The invention thus also relates to a gene therapy method comprising astep of in-vivo or ex-vivo expression of the β2m or of a functionalvariant thereof, as active ingredient. Different types of viral ornon-viral vectors, described in the literature, may be adapted toexpress the β2m protein for this purpose [Urnov et al. (2005) Highlyefficient endogenous human gene correction using designed zinc-fingernucleases, Nature, 435:577-579]. Preferably, the gene therapy vectoraccording to the invention enables the expression in the human body ofthe β2m protein (or of its functional variant) with the exception of anyother active ingredient, and preferably of any other polypeptide.

According to an aspect of the invention, a patient may be treated byperfusion with a solution of liposomes containing the β2m or a vectorexpressing that protein or by transfusion of lymphocytes from patientsplaced in contact with the β2m beforehand. This placing in contact maybe carried out by an “ex vivo” incubation of lymphocytes extracted froma sample of blood taken previously from the same patient.

According to a preferred aspect of the invention, the medicamentcomprising the β2m is prepared in a saline form. A preferred process forpreparing the medicament consists in incubating the β2m in saline form,ex-vivo, in contact with the serum of the patient for whom themedicament is intended.

The pharmaceutical compositions according to the invention describedabove may take any appropriate form known to the person skilled in theart for their oral administration, by injection, perfusion orinhalation.

Another aspect of the invention concerns the diagnosis of autoimmunediseases, more particularly the diagnosis of the diseases cited above,by in-vivo or in-vitro determination of the HC/β2m ratio of the MHC Icomplexes.

The method of diagnosis according to the invention preferably comprisesone or more of the following steps consisting of:

i) taking cells from a patient in whom an autoimmune disease is to bescreened, preferably lymphocytes;

ii) extracting the MHC I complexes from those cells, and if necessary;

iii) determining the respective quantities of HC and of β2m contained insaid complexes;

iv) establishing the HC/β2m molar ratio; and

v) comparing the HC/β2m ratio obtained with the results obtainedpreviously from other patients.

The HC/β2m ratio may be established for the whole of the cell (HC/β2mcell ratio), or, preferably, for the membrane (HC/β2m ratio of themembrane MHC I complexes). Preferably, the method of diagnosis accordingto the invention comprises a step of comparing the HC/β2m ratio withthat of a control, or else in the context of monitoring a patient, withother previously determined ratios.

The respective quantities of the proteins of HC and β2m may bedetermined in standard manner according to the methods known to theperson skilled in the art, for example by quantitative immuno-detection(e.g. ELISA, Immunodot, “Western Blot”, autoantigen microarrays etc.).The extraction of the MHC-I complexes is performed according to theknown protocols of extracting cell and membrane proteins.

The method of diagnosis according to the invention may be implemented inthe context of therapeutic monitoring of patients suffering from variousautoimmune diseases.

The following examples are intended to supplement the description of theinvention without limiting the scope thereof.

Examples 1—Analysis of the Components of the HLA-I Membrane Complex ofthe Lymphocytes in Patients Suffering from Autoimmune Diseases

Without being bound by theory, the inventors have developed the workinghypothesis that an increase in the HC/β2m ratio may result in reactionsof autoimmune type. In particular, the inventors have considered that anexcess of HC, a reduction in β2m, at the level of the MHC-I complexes,or both at the same time, could give rise to a phenomenon of“over-exposure” of “self” to the TcRs. Note that the β2m protectscertain regions of the HCs and specifically determines the presentationof the “non-self” to the CD 8 T-cells [Hill, D. M. et al. (2003), Adominant negative mutant β2-microgobulin blocks the extracellularfolding of major histocompatibility complex class I heavy chain. JBC.278: 5630-5638].

To verify this hypothesis, a first analysis was made to determine themolar quantities of HC and of β2m in the MHC I complexes extracted fromlymphocytes of four patients. The results of these analyses arepresented in Table 1, commented upon above.

The lymphocytes were isolated from the blood of healthy donors and fromthe patients according to the method of Lightbody J. [Manual of ClinicalImmunology, Rose N R., Friedman H. Editors American Society forMicrobiology Washington (DC), 1976, pages 851-857] modified by Hofman F.M. et al. [Ann. J. Clin. Pathol. (1982) 77:710-716]. The MHC-I complexesare detected on the whole lymphocytes or on the plasma membranesprepared according to the method of Warley A. et al. [Biochim.Biophys-Acta (1973), 323: 55-66] with a few modifications. The detectionof the protein components of the MHC-Is was carried out byelectrophoresis (SDS-PAGE system), according to Laemmli U.K. [Nature(1970) 227:680-685] then by electro-transfer onto membranes of PVDF andimmunoblotting according to the method of Towbin H. et al. [Proc. Natl.Acad. Sci. USA (1979) 76:4350-4354]. The revelations were conducted bysecondary antibodies coupled to alkaline phosphatase using an NBT-DCIPmixture.

It was verified that the excess of the heavy chains was indeed ofmembrane origin by isolation of the plasma membranes and use of themethod of binding to glutaraldehyde described later.

As indicated above, four other cases of MS, and two cases of Crohn'sdisease show a cell ratio of HC/β2m>1. These observations incited theapplicant to develop an experimental approach enabling the balance ofHC/β2m (MHC-I) to be restored, in particular by use of liposomes.

2—Preparation of Liposomes Loaded with β2m 2.1—Evaluation of theQuantity of β2m to be Delivered to the Patients

To bring the β2m on the surface of the lymphocytes into excess, itsconcentration in the blood should be increased within “reasonable”limits in order not to trigger the signaling channels on the cellshaving a potential for multiplication.

Given the facility with which β2m detaches from the membranes andcirculates in the blood and renal system, the blood β2m concentrationshould be brought to between 3 mg/l and 8 mg/l (the normal concentrationvaries at around 2 mg/l of blood). This increase leads to the adsorptionof the β2m at the surface of the cells.

Note that the major histocompatibility complexes of type I are composedmole/mole of heavy chains (MW≈43 kDa) and of β2m (MW≈12 kDa). A complex(MW≈55 kDa) is thus composed, by weight, of 79% heavy chain and 21%light chain.

The average protein content of a lymphocyte is 650×10⁻¹² g and theprotein content of its plasma membrane represents only 1% of its totalcontent, i.e. 6.5×10⁻¹² g. If it is considered that the MHC-I onlyrepresents 1% at most of the total content of membrane proteins of aquiescent lymphocyte, the β2m content is thus about 1.4×10⁻¹⁴ g perlymphocyte.

By taking average physiological parameters, 2×10⁶ lymphocytes/ml ofblood and 5 liters of blood per individual, the range in “weight” of thetotal MHC-I/individual (concerning the lymphocytes) would be 1.4×10⁻⁶ gto 1.8×10⁻⁶ g of lymphocyte β2m. These figures are maximum figures inthat our first estimates show values preferentially ranging from0.2×10⁻⁹ to 500×10⁻⁹ g on average in the patients. As the averagequantity in the blood is 10 mg of β2m per individual, i.e. a quantityvery substantially greater than that present on the surface of thelymphocytes, it appears that, in normal conditions, a ratiosubstantially less than 1 already exists between membrane β2m and serumβ2m. It is thus not unreasonable to increase the ratio of β2m in theblood circulation (increasing its oncotic partial pressure) to make upfor the membrane deficit in β2m.

The administration of the β2m may be carried out in two ways:

(1) Administration of liposomes loaded with β2m. This type ofpharmaceutical carrier is current for the administration of peptides,antibodies, genetic material etc. The use of liposomes (“artificial” orsynthetic membranes) promotes the contact between the cell surface andthe active ingredient;

(2) Incubation of the active ingredient in saline form with the serum ofthe patients before administration. The object of this incubation isthat the lipoproteins of the serum act as a vector in the manner ofliposomes.

The degree of incorporation of β2m in the lymphocytes further to theadministration by method 1 or 2 may be compared with a controladministration; in the latter case, the β2m saline perfusion isadministered at 0.10 mg/ml (total volume 150 ml), which provides 3 mg ofβ2m per liter of blood (batch with 15 mg of β2m/150 ml of liposomesolution designated “Batch 15”).

2.2—Formulation of the Liposomes

For the preparation of the liposomes (for 1 ml): after evaporation ofdichloromethane (CH₂Cl₂) containing the constituents to dryness undernitrogen, a film containing the phosphatidylcholine, with or withoutaddition of cholesterol, with or without addition of sphingomyelin orwith addition of cholesterol and sphingomyelin is constituted. For thethree compounds (phosphatidylcholine, cholesterol and sphingomyelin) theproportions are respectively 10 M, 2 M and 1 M—i.e. for 1 ml of finalsolution 7.60 mg, 0.76 mg and 0.38 mg. To this film there is added 1 mlof a saline solution (PBS 10 mM. pH=7.4; HANKS, Tris/Glycine or DMEM)containing 2 mg of β2m. The molarity remains the same for each of thecompounds If liposomes made from phosphatidylcholine (10 M), fromphosphatidylcholine (10 M) and from cholesterol (2 M), fromphosphatidylcholine (10 M) and from sphingomyelin (1 M) are produced.However other molarities concerning the lipid components may be used.The quantities of proteins may be different and the pH may be greaterthan 7.4 depending on the case. The dispersion of the lipid film iscarried out by stirring up to 3 hours at a temperature between 20 and37° C.

The liposomes are formed by the so-called “detergent/dialysis” method,or else by the so-called “extrusion” method. For the latter, thesolution (Lipofast®, Sodexim S. A., 51140 Muizon, France) is passed 41times through filter membranes of 100 nm in polycarbonate under apressure of 69 bars. The liposomes obtained are of homogenous size. Theliposomes, in this case, are kept for 2 days at 4° C. and added to thelymphocyte suspension (diameter<100 nm; FIG. 4). On larger scale, thesolution (31/hr; sodexim 2770; emulsifflex c3; sodexim s.a.) is thenpassed 4 times at a pressure of 450 bars to obtain SUVs (smallunilamellar vesicules).

In “pre-pilot” assays, in order to show the incorporation of the β2m inliposomes, the adsorption of the liposomes on the cell surface and thetransfer of the protein from the liposome to the inside of the cells, weproduced fluorescent liposomes. According to the assays, liposomes wereprepared which fluoresce at 520 nm or 572 nm. For this, 0.5 M of NBD-PC(1-oleyl-2-(-6-(((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)hexanoyl)-sn-glycero-3-phosphocholine)(excitation at 490 nm and emission at 520 nm) or 0.5 M of Liss Rhod PE(1,2-dioleyl-sn-glycero-3-phosphatidyletholamine-N-(lissamine rhodamineB sulfonyl)(ammonium salt) (excitation at 541 nm and emission at 572 nm)were added to the lipid mixture before evaporation and obtainment of thelipid film (see above).

At pre-pilot scale, the liposomes were produced by thedetergent/dialysis method. By this technique, well-calibrated and stableSUVs were also obtained.

Briefly, after stirring up to 3 hours at a temperature between 20 and37° C., the micellar suspension is dialyzed against a saline solutioncontaining β2m as well as 4 μM (0.8 mg/ml) of n-hexyl-βD-glucopyranosidefor 12 h at 4° C. in a microdialysis apparatus. The dialysis membraneshave an cut-off of 3.5 kDa and the n-hexyl-βD-glucopyranoside(detergent) is diluted to least 1 ppm in the final solutions.

The liposomes obtained have a size of approximately 200 nm diameter.They are stable over 3 months, at least, at ambient temperature andcontain at least 0.1 mg (β2m)/ml of initial solution.

2.3—Application of Test Liposomes onto Lymphocytes Maintained “Ex Vivo”in Culture

To show the relevance of formulating the β2m protein for the purpose oftargeting the MHC complexes of the lymphocytes, in the form of liposomesuspensions, lymphocytes were incubated with liposomes loaded withalbumin, a protein that is possible to detect by fluorescence using arelatively simple technique.

The incorporation of the protein into the liposomes and the applicationof the liposomes produced according to the protocol described above onthe basis of phosphatidylcholine with Liss Rhod PE were tested ex-vivo.

The protein was rendered fluorescent by marking with fluorescamine, acompound whose fluorescence is comparable with that of DAPI (Di AminoPhenyl Indol; excitation at 372 nm and emission at 456 nm). The albumincrystallized from bovine serum was rendered fluorescent using binding bycovalency of the fluorescamine on the N-terminal end of the protein,using the method described by Hames B. D. et al. [Gel Electrophoresis ofProteins, a practical approach, Hames B D. and Rickwood D. eds. Thepractical Approach Series, 2^(nd) Edition. IRL Press, Oxford, New York,Tokyo. p. 67] except that the marking is carried out in a Tris-Hcl (25mM)/Glycine (192 mM) (pH=8.3) buffer not containing detergent (SDS). Theliposomes formed, as described earlier, contain 2 mg of fluorescentalbumin per ml of liposome solution.

Next, 0.2 ml of a suspension of lymphocytes (Hank's/0.5 mM EDTA,pH=7.4), containing 250 000 lymphocytes, was incubated with 0.2 ml ofthe liposomes so formed containing albumin (Buffer 25 mM Tris-Hcl/192 mMGlycine, pH=8.3) for 1 hour at 37° C. in a moist atmosphere saturatedwith CO₂ (5%).

Finally, control liposomes (200 μl), control lymphocytes (200 μl) andlymphocytes treated by liposomes loaded with protein (200 μl) weresedimented on coverslips treated and covered with polylysine, andlaminin, using a method described by Rakotoarivelo C et al. [Receptorsto steroid hormones and aromatase are expressed by cultured motoneuronsbut not by glial cells derived from rat embryo spinal cord (2004)Neuroendocrinology 80:284-297].

The preparations were observed directly by epifluorescence microscope(Axiovert; Zeiss, Germany) or fixed with a solution at 4% (plv) ofparaformaldehyde in water for 30 minutes, the coverslips being rinsed 3times with PBS (160 mM, pH=7.2). The preparations were gently renderedpermeable, with 0.1% of Triton X-100 (v/v), prepared in PBS, for 5 min.

The cells were marked by primary anti HLA-ABC antibodies. In certaincases, the cell nuclei were marked with Hoechst (fluorescence 450 nmblue emission, DAPI). The primary antibodies, produced in rabbits, arethe same as those used for the “Western blot”. The primary antibodiesare diluted to 1/50 and the secondary antibodies bound to FITC (greenfluorescence) and produced in the goat and are diluted to 1/160. Theincubations of the antibodies were carried out in PBS containing 2%bovine serum Albumin. For the ×63 lens, the coverslips were mounted withFluorsave (Calbiochem, USA).

The photographs of FIGS. 2 and 3 show that the liposomes are adsorbed onthe surface of the lymphocytes and that the marked protein, contained inthe liposomes, is deposited on the membrane surface of said lymphocytes.

The results clearly demonstrate the feasibility of the experimentalapproach that we propose to restore the HC/β2m membrane equilibrium.

Other liposomes with green fluorescence were also produced according tothe extrusion method containing human β2m purified from urine (Sigma,USA) at a concentration of 0.6 mg/ml. The β2m was marked with rhodamineβ isothiocyanate, which has red fluorescence (excitation: 540 nm;fluorescence: 625 nm). The β2m—Rhodamine B binding was made using themethod described by Riggs et al. [(1958) Am. J. Pathol. 34: 1081-1097].After binding, the protein was purified in a Sephadex column (Pharmacia,Sweden, G-10: bed volume 9 ml; inside diameter of the column 0.7 mm).The column is hydrated in PBS (Bio-rad, 10 mM phosphates, 150 mM NaCl,pH=8.3). The protein was eluted (4.5-7.0 ml) in PBS diluted 1:1 inmilli-Q H₂O. The liposomes formed (FIG. 4) were purified in a similarcolumn (2.5-6.5 ml) and concentrated twice with a rotavapor (Buchi,Switzerland). The lymphocytes isolated from the blood of patient P1 areincubated as described earlier for 90 minutes and observed with afluorescence microscope. The results show an incorporation of the β2m in89% of the lymphocytes (FIG. 5).

2.4—Stability of Liposomes (FIGS. 6 to 8)

The “test” liposomes obtained above containing albumin were tested invarious conditions in order to evaluate their stability over time.

The stability was tested on batches 30 (corresponding to 30 mg ofAlbumin for 150 ml of liposome) and 60 (corresponding to 60 mg ofAlbumin for 150 ml of liposome) against time and incubation temperature.

The lipids constituting the liposomes were composed of 636 nmol of PCand 31.8 nmol of NBD-PC-Oleyl. After evaporation to dryness under astream of nitrogen, the mixture of lipids is solubilized drop by dropwith strong stirring with 1 ml of PBS (pH adjusted to 7.2) containing200 or 400 μg of albumin (Sigma, USA) (batches 30 and 60, respectively).Next, the liposomes were obtained by mechanical extrusion with theLiposofast-basic system (Sodexim, France). Each batch was then purifiedin a Sephadex G10 column. At a set time, 50 μl of each batch wasdeposited on a poly-D-lysine/laminine-coated coverslip and incubated at37° C. for 12 h. Next, the biological material was bound by usingglutaraldehyde for 30 at 4° C. The images (stability at 1 month ofstorage, FIG. 6) were taken using the Axiovert 200 (Zeiss)epifluorescence microscope and recorded with the Axion vision softwareapplication. Regarding the statistical studies (FIG. 7), the diameter ofthe liposomes was measured using Serf software (http://www.org/serf).For the study of each batch, the liposome population was divided intothree classes: <50, between 50 and 100 nm and >100 nm diameter. Theheights of bar charts represent the percentage of each sizesub-population. The batch 60 kept at 37° C. shows the greatest stabilityfor 60 days of storage: 95% of the liposomes have a diameter <100 nm,which is an ideal diameter for the transfer of protein to the cellsurface.

As for batch 80 (80 mg of β2m; FIG. 8), this was tested for storage at25° C. in order to minimize contamination and evaporation, for 6 and 40days. The preparation method was the same as the previous one forbatches 30 and 60 (albumin) except that non-fluorescent β2m was used(533 μg/ml of PBS) and the purification was carried out by dialysiscassette (membrane with 20 kDa cut-off, Thermo Scientific, USA). The barchart of FIG. 8 clearly shows that at this stage of storage 98% of theliposomes maintain the ideal size, and this during up to 40 days, forthe transfer of β2m to lymphocytes (i.e. diameter<100 nm).

2.5—Protection of the Exogenous β2m Conferred by the Liposomes AgainstProteolytic Degradation by Human Sera (FIG. 9)

Sera were taken from healthy donors and donors suffering from autoimmunediseases (Hashimoto's thyroiditis, rheumatoid polyarthritis). These serawere incubated (90 μl) for 15 days at 25° C. in the presence of 2 μg ofpure β2m (Sigma-Aldrich, USA) or in liposome form (Batch 30 liposomescorresponding to 30 mg of β2m per 150 ml of liposomes). The totalreaction volume was 130 μl, completed if necessary with PBS (sodiumphosphate 10 mM, sodium chloride 150 mM, pH=7.2). 10 μl from thatreaction medium (corresponding to 150 ng), completed to 30 μl withdenaturing buffer (SDS-PAGE, Laemmli) containing 6 M of urea, wassuccessively removed at 0, 1, 2, 3, 6, 10 and 15 days. These sampleswere kept at −20° C. until analysis.

After collecting all the samples, these were incubated for 1 h at 50° C.Next, the proteins were separated by SDS-PAGE on 12% acralamide gel (%T=12, % C=2.6) containing 4 M of urea.

After electro-elution on a polyvinylidene difluoride (PVDF) membrane,the presence of β2m was detected by immunoblotting and the intensity ofthe corresponding band was quantified with ImageJ (NIH, USA). By the useof a standard curve, the number of pixels so obtained was converted intopmoles of β2m (10⁻¹² moles). The graphs presented in FIGS. 9 A to Frepresent an example of results obtained and express the quantity of β2m(in pmoles) over time. It can be noted that in the persons sufferingfrom autoimmune diseases, the free β2m added to the serum is degradedover time, which is not the case in the control.

In conclusion, there is a progressive and significant degradation of thefree β2m by the serum of autoimmune patients which is not found in thecontrol. On the other hand, this degradation is not observed when theβ2m is encapsulated in liposomes. To be precise, the liposomes appear toprotect the β2m against degradation by serum, since no significantreduction in the quantity was observed.

In conclusion, the liposomes protect the β2m from the degradation byserum.

2.6—Association of the β2m Contained in the Liposomes with the HLA IHeavy Chains Located on the Membrane Surface (FIG. 10).

In a healthy person and in physiological conditions, the molecules ofβ2m expressed on the lymphocyte surface are bound non-covalently to theHLA heavy chains with a ratio of 1:1.

In order to view and quantify these protein associations, we developed atechnique enabling those proteins, among which the HLA-β2m dimers, to belinked together covalently

The development of this technique was necessary to calculate the exactmembrane HC/β2m ratio and to show that the addition of β2m in liposomeform specifically associates with the heavy chains of HLA-I.

For this we exploited the capacity of a dialdehyde, glutaraldehyde, tobind the amine groups of the proteins by its two aldehyde groups. Thesealdehyde groups are linked together by a flexible chain of threemethylenes which enables glutaraldehyde to statistically crosslink twoamine groups coming from two interacting proteins (Sun, T. T., et al.(1974) Protein-protein proximity in the association of ribosomalsubunits of Escherichia coli: crosslinking of 30S protein S16 to 50Sproteins by glutaraldehyde or formaldehyde. J. Mol. Biol. 87(3):509-22).

According to this procedure, 5 million lymphocytes purified by MSL werewashed once with PBS (pH=7.2) to eliminate possible traces of freeamines, then pelleted by centrifugation at 10 000 g for 10 min and thesupernacent liquid eliminated. The cells were then incubated for 5minutes at ambient temperature in 1 ml of PBS containing 0.25% ofglutaraldehyde. During this incubation, the tube was inverted severaltimes.

The cessation of the reaction was obtained by the addition of 100 μl oftris 1 M (pH=7.2), the excess amine groups provided by the Tris bufferneutralizing the glutaraldehyde. The lymphocytes were retrieved bycentrifugation at 10 000 g for 10 min, then washed in 1 ml of PBS inorder to eliminate traces of glutaraldehyde. After centrifugation, thepellet was retrieved in 400 μl of Laemmli buffer containing 4 M of urea,comprising antiproteases (Roche Diagnostics GmBH, Germany) and 5% ofβ-mercaptoethanol, then kept at −20° C. until analysis.

In order to ensure the lysis of the cells, the sample underwent 3 cyclesof freezing-thawing and was extensively vortexed. The samples were thenincubated for 5 min at 95° C. and centrifuged for 10 min at 4000 g toeliminate any insoluble residue. The proteins (10 μl of homogenatecorresponding to 125 000 lymphocytes) were separated by SDS-PAGE onacrylamide gels of 10% (% T=10, % C=2.6) containing 4 M urea at constantvoltage (120V). The proteins so separated were transferred semi-dry for40 min at 13 V in the presence of tris-glycine buffer with 10% methanol,on a PVDF membrane activated beforehand with methanol.

The detection of the β2m was carried out by incubation at ambienttemperature for 1 hour with a primary anti-β2m antibody diluted to 1/600(DakoCytomation, Denmark) then again 1 hour with a secondary anti-rabbitantibody coupled to alkaline phosphatase and diluted to 1/20 000(Sigma-Aldrich, USA). The quantification of the intensity of the bandsobtained was performed with the ImageJ software application (NIH, USA).

FIG. 10A shows a photograph of the gel obtained. In the presence ofglutaraldehyde, a band at 55 kDa is visible in addition to the usualband at 12 kDa. This band at 55 kDa corresponds to the HLA-β2m complex,the molecular weight corresponding to the addition of the molecularweights of a β2m molecule and of a heavy chain: 12+43 kDa=55 kDa. On thesame lane, the band at 12 kDa corresponds to the free non-complexed β2m.After quantifying the intensity of each band, it was checked that thecumulative intensity of these two bands at 12 and 55 kDa corresponds tothe intensity of the band at 12 kDa in the lane “without glutaraldehyde”and which corresponds to the total β2m. In both lanes, with and withoutglutaraldehyde, the same quantity of total proteins corresponding to thesame number of lymphocytes was deposited.

In order to provide evidence that the quantification of the band at 55kDa does indeed give the quantity of HLA-β2m complex present on thelymphocyte surface, a technique was used in parallel to purify plasmamembranes from lymphocytes. This technique specifically allowed us tostudy proteins of the lymphocyte plasma membrane. The presence ofmembrane β2m was determined and quantified (see FIG. 10B). The resultobtained is comparable to the quantification of the band at 55 kDa,which confirms that this band does indeed correspond to the membraneHLA-β2m complex. Furthermore, the proportion of membrane β2m relative tothe total β2m (FIG. 10B) is equal to the ratio of intensities of the 55kDa band/total β2m 12 kDa band (FIG. 10A).

The glutaraldehyde technique thus validated, was used to view andquantify the degree of incorporation on the lymphocytes isolated fromhuman blood, of the β2m conveyed by liposomes.

Two donors, one healthy, used as a control, and the other suffering frommultiple sclerosis, were selected. The second donor was chosen onaccount of his deficit in membrane β2m relative to the heavy chains ofHLA-I. This patient has a membrane HC/β2m ratio equal to 1.7 which meansthat the lymphocyte membrane contains 69% more heavy chains than β2m.

The lymphocytes from the two donors (25 ml blood) were separated in twobatches of 4 ml each; 2 ml of liposomes containing β2m at aconcentration of 40 mg for 150 ml (Batch 40), were added to the 4 ml oflymphocytes. The T₀ lymphocytes were immediately collected and washedwith PBS. The lymphocytes sampled at T₉₀, were incubated with theliposomes for 90 min. at 37° C. before being collected and washed withPBS in order to eliminate the excess liposomes not having reacted.

In each of the two conditions, we analyzed two populations oflymphocytes that had or had not been treated with glutaraldehyde (seeprotocol above)

The total proteins, in each condition, were separated by SDS-PAGE, thenrevealed by western blot. The results obtained are illustrated in FIG.10C and we found that after quantification, contrary to the control, andin presence of glutaraldehyde, the intensity of the 55 kDa band(representing the HLA-β2m complex) had increased by 55% at T₉₀ relativeto T₀.

This increase is consistent with the deficit in β2m in that patient,which cause the presence of free HLA-1 chains on the lymphocyte surface.Thus the heavy chains do indeed associate with the exogenous β2mprovided by the liposomes.

The experimental approach implemented enabled the proof to be providedfor the therapeutic concept of the invention, i.e. that:

-   -   It is possible to reestablish the HLA-β2m balance by an addition        to the lymphocyte surface of exogenous β2m in the form of        liposomes.    -   A patient having a deficit of β2m may incorporate more β2m than        a control who has no need for it.    -   The incorporated β2m does indeed associate with the free HLA        molecules to form HLA-β2m dimers.

In summary, the experimental data obtained confirm that it is possible,using liposome preparations of β2m, to target lymphocytes presentingfree heavy chains (HC/β2m>1) for the purpose of reestablishing a HC/β2mratio close to the physiological norm, i.e. approaching 1.

3—Toxicity Analysis of Liposome Compositions of β2m

The β2m in liposome form was tested “in vitro” for its possible toxicityon cultures of liver, kidney, skeletal muscle and heart cells of humanorigin.

3.1—Types of Cells Tested

The cells tested and the culture media were purchased from ScienceIIResearch Laboratories (6076 Corte Del Cedro, Carlsbad, Calif.).

a. HCF: Primary human cardiac fibroblast cells, batch No. 2136 Culturemedium: FM (Fibroblast Medium), batch No. 5673+Fibroblast GrowthSolution, batch No. 5863+FBS 10%+penicillin solution (100U/ml)-Streptomycin (100 μg/ml), batch No. 5917

b. HREpiC: primary human renal epithelial cells, batch No. 0546 Culturemedium: Epithelial Cells Medium, batch No. 5967+Epithelial Cells GrowthSolution, batch No. 5855+FBS 10%+PS

c. HH: primary human hepatocyte cells, batch No. 4607 Culture medium: HM(Hepatocyte Medium), batch No. 5933+Hepatocyte Growth Solution, batchNo. 5722+FBS 10%+PS

d. HSkMC: primary human skeletal muscle cells, batch No. 5606 Culturemedium: Skeletal Muscle Cells Medium+SkMGS+FBS 10%+PS

The culture flasks or dishes were placed in an incubator (Sanyo) at 37°C., 5% CO₂ and with saturated humidity, (bath containing ultra-purewater filtered with 0.22 μm, Nanopure, Thermo-Fisher).

The culture substrate for the primary human cells is cell culturetreated plastic (TPP, Switzerland) incubated with poly-L-lysine at 2μg/cm² (Clinisciences; ScienceII Research Laboratories, batch No. 5826,solution: 10 mg/ml) for one night in the incubator and rinsed twice withsterile ultra-pure water before inoculation.

3.2—Detachment and Dissociation of the Cell Layer

The detachment of the cell layer was carried out by eliminating theprepared medium from the culture flask then by rinsing the layer withsterile PBS (SIGMA, batch No. 088K2356) then by treating it with asolution of 0.05% trypsin (SIGMA Trypsin Ref T-1426, batch No.020M7354), EDTA 0.2 g, NaCl 8 g, KCl 0.4 g, NaHCO3 0.58 g, Glucose 1 g(SIGMA), qs 1 liter ultra-pure water, solution sterilized by membranefiltration (PES) of 0.22 p porosity, CML batch No. 668919), the volumeof the trypsin solution was adjusted to the type of flask (e.g. 1 ml fora flask of 25 cm²), then the culture flask was placed at 37° C. (Sanyoincubator) for three to four minutes.

When the cells were detached from their substrate the dissociation wasimplemented in the presence of culture medium with serum (inhibition ofthe enzyme action of the trypsin) sent to and from in a pipette (from 5to 10 ml according to the cell type).

3.3 Toxicity Test

The cells were counted using a Thoma cell (Thermo Fisher) under anoptical microscope (Nikon) and were seeded in an amount of 5000 cellsper well in 200 μl of their respective culture medium in a flat bottomedculture dish with 96 wells of cell culture treated plastic (NUNC, batchNo. 114754) then after preparation the dishes were placed in anincubator for 24 h. The various dilutions of the substances to test wereconcentrated three times in 100 μl of medium without antibiotics whichwere added to the 200 μl of each well to treat (total volume: 300 μl).At 24 h, at 48 h and 72 h, the treated wells and the control wells wereexamined in accordance with the protocols for the MTT (Thiazolyl BlueTetrazolium Bromide) [Liu Y. et al. (1997) Mechanism of cellular MTTreduction. J. Neurochem. 69: 581-593] and for the dosage of the proteins(Ref 23227, BCA protein Assay kit; Pierce) to evaluate the celltoxicity:

-   -   Addition of MTT solution for final concentration 25 μg/mL    -   Incubation 1 h at 37° C.    -   Aspiration of the medium    -   Addition of 100 μL DMSO (200 μL if saturation DO)    -   Reading of the dish (Biorad) at 490 nm    -   Computer processing with Excel.    -   Subtraction of the background noise using empty wells (blanks)    -   Determine the ratio of DO X wells/DO control wells    -   Trace the curve of that ratio against the drug concentration

The toxicity was measured at 24 h and 48 h of treatment i.e. t₀+48 h andt₀+72 h

FIGS. 11 and 12 clearly show that, even at a high dose (Batch 132), theliposome-coated β2m does not affect the viability of the hepatocytes andkidney cells, which are however sensitive to β2m. The same applies forthe cells of cardiac origin and skeletal muscle cells (results notshown).

1-21. (canceled)
 22. A pharmaceutical product, characterized in that itconsists of β2-microglobulin or of a functional variant of that proteinpresenting at least 70% identity with the human β2-microglobulinprotein, in a pharmaceutically acceptable carrier.
 23. A pharmaceuticalproduct according claim 22, wherein said active ingredient is the humanβ2-microglobulin protein.
 24. A pharmaceutical product according toclaim 22, wherein said active ingredient is a functional variant of theβ2-microglobulin protein presenting at least 80%, and preferably 90%identity with the human β2-microglobulin protein.
 25. A method oftreating an autoimmune disease, comprising administering to a subject inneed thereof an effective amount of the pharmaceutical product accordingto claim
 22. 26. The method according to claim 25, characterized in thatthe autoimmune disease treated is rheumatoid polyarthritis, systemiclupus erythematosus, Sjögren's syndrome, scleroderma, fibromyalgia,myositis, ankylosing spondylitis, insulin dependent diabetes of type I,Hashimoto's thyroiditis, Addison's disease, Crohn's disease, Celiac'sdisease, multiple sclerosis or amyotrophic lateral sclerosis.
 27. Themethod according to claim 25, wherein the autoimmune disease treated isamyotrophic lateral sclerosis (ALS).
 28. The method according to claim25, wherein the autoimmune disease treated is multiple sclerosis. 29.The method according to claim 25, wherein the autoimmune disease treatedis Crohn's disease.
 30. The method according to claim 25, wherein theautoimmune disease treated is rheumatoid polyarthritis.
 31. The methodaccording to claim 25, wherein the autoimmune disease treated is insulindependent diabetes of type I.
 32. A method of increasing the ratio ofblood β2-microglobulin to a concentration comprised between 2.5 and 12mg/l, preferably between 3 and 8 mg/l, more preferably between 3 and 5mg/l in a patient suffering from an auto-immune disease, comprisingadministering to said patient an effective amount of the pharmaceuticalproduct according to claim
 22. 33. A method of restoring a normalHC/β2-microglobulin molar ratio within the membrane MHC-I complexes in apatient suffering from an auto-immune disease, comprising administeringto said patient an effective amount of the pharmaceutical productaccording to claim
 22. 34. A method of preventing a β2-microglobulindeficit from occurring in the MHC-I complexes in a patient sufferingfrom an auto-immune disease, comprising administering to said patient aneffective amount of the pharmaceutical product according to claim 22.35. A pharmaceutical product according claim 22, characterized in thatit consists of a liposome loaded with β2-microglobulin or with afunctional variant of that protein.
 36. A pharmaceutical productaccording to claim 22, characterized in that β2-microglobulin isprepared in saline form and incubated beforehand ex-vivo in contact withthe blood, the serum or the lymphocytes of the patient to treat.
 37. Acomposition comprising a pharmaceutical product according to claim 22.38. A method of diagnosis of an autoimmune disease, characterized inthat it comprises a step consisting of determining the intracellular ormembrane HC/β2-microglobulin ratio of the MHC-I complexes in a patient.39. A method of diagnosis according to claim 38, characterized in thatthe HC/β2-microglobulin ratio of the MHC-I complexes is a membraneratio.
 40. A method according to claim 39, characterized in that itcomprises the steps of: i) taking cells from a patient in whom anautoimmune disease is to be screened, preferably lymphocytes; ii)extracting the MHC-I complexes from those cells; iii) determining therespective quantities of HC and of β2-microglobulin contained in saidMHC-I complexes; iv) establishing the HC/β2-microglobulin molar ratio ofsaid MHC-I complexes; and v) comparing the HC/β2-microglobulin ratioobtained with that of a control sample.